RESUMO
BACKGROUND: Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1ß. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury. METHODS: After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing. RESULTS: In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4-L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1ß. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels. CONCLUSIONS: Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well.
Assuntos
Inflamassomos , Traumatismos dos Nervos Periféricos , Humanos , Inflamassomos/metabolismo , Neurônios Motores/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças Neuroinflamatórias , Nervo Isquiático/lesõesRESUMO
Age-related blood-brain barrier (BBB) disruption and cerebromicrovascular rarefaction contribute importantly to the pathogenesis of both vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). Recent advances in geroscience research enable development of novel interventions to reverse age-related alterations of the cerebral microcirculation for prevention of VCID and AD. To facilitate this research, there is an urgent need for sensitive and easy-to-adapt imaging methods that enable longitudinal assessment of changes in BBB permeability and brain capillarization in aged mice and that could be used in vivo to evaluate treatment efficiency. To enable longitudinal assessment of changes in BBB permeability in aged mice equipped with a chronic cranial window, we adapted and optimized two different intravital two-photon imaging approaches. By assessing relative fluorescence changes over the baseline within a volume of brain tissue, after qualitative image subtraction of the brain microvasculature, we confirmed that, in 24-mo-old C57BL/6J mice, cumulative permeability of the microvessels to fluorescent tracers of different molecular masses (0.3 to 40 kDa) is significantly increased compared with that of 5-mo-old mice. Real-time recording of vessel cross-sections showed that apparent solute permeability of single microvessels is significantly increased in aged mice vs. young mice. Cortical capillary density, assessed both by intravital two-photon microscopy and optical coherence tomography was also decreased in aged mice vs. young mice. The presented methods have been optimized for longitudinal (over the period of 36 wk) in vivo assessment of cerebromicrovascular health in preclinical geroscience research.NEW & NOTEWORTHY Methods are presented for longitudinal detection of age-related increase in blood-brain barrier permeability and microvascular rarefaction in the mouse cerebral cortex by intravital two-photon microscopy and optical coherence tomography.
Assuntos
Envelhecimento/patologia , Barreira Hematoencefálica/diagnóstico por imagem , Permeabilidade Capilar , Córtex Cerebral/irrigação sanguínea , Microscopia Intravital , Microscopia de Fluorescência por Excitação Multifotônica , Rarefação Microvascular , Microvasos/diagnóstico por imagem , Tomografia de Coerência Óptica , Fatores Etários , Envelhecimento/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Masculino , Camundongos Endogâmicos C57BL , Densidade Microvascular , Microvasos/metabolismo , Microvasos/patologia , Fatores de TempoRESUMO
Spreading depolarization (SD) is a wave of mass depolarization that causes profound perfusion changes in acute cerebrovascular diseases. Although the astrocyte response is secondary to the neuronal depolarization with SD, it remains to be explored how glial activity is altered after the passage of SD. Here, we describe post-SD high frequency astrocyte Ca2+ oscillations in the mouse somatosensory cortex. The intracellular Ca2+ changes of SR101 labeled astrocytes and the SD-related arteriole diameter variations were simultaneously visualized by multiphoton microscopy in anesthetized mice. Post-SD astrocyte Ca2+ oscillations were identified as Ca2+ events non-synchronized among astrocytes in the field of view. Ca2+ oscillations occurred minutes after the Ca2+ wave of SD. Furthermore, fewer astrocytes were involved in Ca2+ oscillations at a given time, compared to Ca2+ waves, engaging all astrocytes in the field of view simultaneously. Finally, our data confirm that astrocyte Ca2+ waves coincide with arteriolar constriction, while post-SD Ca2+ oscillations occur with the peak of the SD-related vasodilation. This is the first in vivo study to present the post-SD astrocyte Ca2+ oscillations. Our results provide novel insight into the spatio-temporal correlation between glial reactivity and cerebral arteriole diameter changes behind the SD wavefront.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical , Oscilometria , Animais , Arteríolas/metabolismo , Astrócitos/citologia , Circulação Cerebrovascular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Neurônios , Córtex Somatossensorial/metabolismo , VasodilataçãoRESUMO
Triple negative breast cancer presents higher mortality and poorer survival rates than other breast cancer (BC) types, due to the proneness to brain metastases formation, which are usually diagnosed at advanced stages. Therefore, the discovery of BC brain metastases (BCBM) biomarkers appears pivotal for a timely intervention. With this work, we aimed to disclose microRNAs (miRNAs) and extracellular vesicles (EVs) in the circulation as biomarkers of BCBM formation. Using a BCBM animal model, we analyzed EVs in plasma by nanoparticle tracking analysis and ascertained their blood-brain barrier (BBB) origin by flow cytometry. We further evaluated circulating miRNAs by RT-qPCR and their brain expression by in situ hybridization. In parallel, a cellular model of BCBM formation, combining triple negative BC cells and BBB endothelial cells, was used to differentiate the origin of biomarkers. Established metastases were associated with an increased content of circulating EVs, particularly of BBB origin. Interestingly, deregulated miRNAs in the circulation were observed prior to BCBM detection, and their brain origin was suggested by matching alterations in brain parenchyma. In vitro studies indicated that miR-194-5p and miR-205-5p are expressed and released by BC cells, endothelial cells and during their interaction. These results highlight miRNAs and EVs as biomarkers of BCBM in early and advanced stages, respectively.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , MicroRNA Circulante/sangue , Vesículas Extracelulares/patologia , Animais , Barreira Hematoencefálica , Neoplasias Encefálicas/secundário , Neoplasias da Mama/genética , Linhagem Celular Tumoral , MicroRNA Circulante/genética , Endotélio Vascular/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
By upregulation of cell adhesion molecules and secretion of proinflammatory cytokines, cells of the neurovascular unit, including pericytes and endothelial cells, actively participate in neuroinflammatory reactions. As previously shown, both cell types can activate inflammasomes, cerebral endothelial cells (CECs) through the canonical pathway, while pericytes only through the noncanonical pathway. Using complex in vitro models, we demonstrate here that the noncanonical inflammasome pathway can be induced in CECs as well, leading to a further increase in the secretion of active interleukin-1ß over that observed in response to activation of the canonical pathway. In parallel, a more pronounced disruption of tight junctions takes place. We also show that CECs respond to inflammatory stimuli coming from both the apical/blood and the basolateral/brain directions. As a result, CECs can detect factors secreted by pericytes in which the noncanonical inflammasome pathway is activated and respond with inflammatory activation and impairment of the barrier properties. In addition, upon sensing inflammatory signals, CECs release inflammatory factors toward both the blood and the brain sides. Consequently, CECs activate pericytes by upregulating their expression of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome-forming pattern recognition receptor. In conclusion, cerebral pericytes and endothelial cells mutually activate each other in inflammation.
Assuntos
Encéfalo/patologia , Comunicação Celular , Células Endoteliais/patologia , Inflamassomos/metabolismo , Pericitos/patologia , Transdução de Sinais , Animais , Inflamação/metabolismo , Inflamação/patologia , Suínos , Junções Íntimas/metabolismoRESUMO
Spontaneous, recurrent spreading depolarizations (SD) are increasingly more appreciated as a pathomechanism behind ischemic brain injuries. Although the prostaglandin F2α - FP receptor signaling pathway has been proposed to contribute to neurodegeneration, it has remained unexplored whether FP receptors are implicated in SD or the coupled cerebral blood flow (CBF) response. We set out here to test the hypothesis that FP receptor blockade may achieve neuroprotection by the inhibition of SD. Global forebrain ischemia/reperfusion was induced in anesthetized rats by the bilateral occlusion and later release of the common carotid arteries. An FP receptor antagonist (AL-8810; 1 mg/bwkg) or its vehicle were administered via the femoral vein 10 min later. Two open craniotomies on the right parietal bone served the elicitation of SD with 1 M KCl, and the acquisition of local field potential. CBF was monitored with laser speckle contrast imaging over the thinned parietal bone. Apoptosis and microglia activation, as well as FP receptor localization were evaluated with immunohistochemistry. The data demonstrate that the antagonism of FP receptors suppressed SD in the ischemic rat cerebral cortex and reduced the duration of recurrent SDs by facilitating repolarization. In parallel, FP receptor antagonism improved perfusion in the ischemic cerebral cortex, and attenuated hypoemic CBF responses associated with SD. Further, FP receptor antagonism appeared to restrain apoptotic cell death related to SD recurrence. In summary, the antagonism of FP receptors (located at the neuro-vascular unit, neurons, astrocytes and microglia) emerges as a promising approach to inhibit the evolution of SDs in cerebral ischemia.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Circulação Cerebrovascular/efeitos dos fármacos , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Dinoprosta/análogos & derivados , Animais , Isquemia Encefálica/fisiopatologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Infarto Cerebral/tratamento farmacológico , Circulação Cerebrovascular/fisiologia , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Dinoprosta/farmacologia , Masculino , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/fisiopatologia , Prostaglandinas/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Species in the Juncaceae accumulate different types of secondary metabolites, among them phenanthrenes and 9,10-dihydrophenanthrenes in substantial amounts. These compounds have chemotaxonomic significance and also possess interesting pharmacological activities. The present study has focused on the isolation, structure determination, and pharmacological investigation of phenanthrenes from Juncus gerardii. Twenty-six compounds, including 23 phenanthrenes, have been isolated from a methanol extract of this plant. Twelve compounds, the phenanthrenes gerardiins A-L (1-12), were obtained as new natural products. Eleven phenanthrenes [effusol (13), dehydroeffusol (14), effususin A (15), compressin A, 7-hydroxy-2-methoxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene, juncusol, 2-hydroxy-7-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-5-formyl-1-methyl-9,10-dihydrophenanthrene, effususol A, 2,7-dihydroxy-5-hydroxymethyl-1-methyl-9,10-dihydrophenanthrene, and jinflexin C], 1-O-p-coumaroyl-3-O-feruloyl-glycerol, and the flavones apigenin and luteolin were isolated for the first time from this plant. The cytotoxicity of the 23 isolated phenanthrenes in both mouse (4T1) and human (MDA-MB-231) triple-negative breast cancer cells and in a nontumor (D3, human cerebral microvascular endothelial) cell line was tested using an MTT viability assay. The results obtained showed that the dimeric compounds gerardiins I (9), J (10), K (11), and L (12), derived biogenetically from effusol and dehydroeffusol, were cytotoxic to both tumor and nontumor cell lines, while the monomeric compounds exerted no or very low cytotoxicity. Impedance measurements were consistent with the results of the MTT assays performed.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Fenantrenos/química , Fenantrenos/farmacologia , Plantas Tolerantes a Sal/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Magnoliopsida , Camundongos , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Sais de Tetrazólio , TiazóisRESUMO
Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood-brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia-like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co-culturing melanoma cells with cerebral endothelial cells, we observed N-cadherin enrichment at melanoma-melanoma and melanoma-endothelial cell borders. However, for breast cancer cells N-cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Córtex Cerebral/patologia , Melanoma Experimental/patologia , Neoplasias Cutâneas/patologia , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Expressão Gênica , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Transplante de Neoplasias , Especificidade de Órgãos , Cultura Primária de Células , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Células Tumorais CultivadasRESUMO
With age, our cognitive skills and abilities decline. Maybe starting as an annoyance, this decline can become a major impediment to normal daily life. Recent research shows that the neurodegenerative disorders responsible for age associated cognitive dysfunction are mechanistically linked to the state of the microvasculature in the brain. When the microvasculature does not function properly, ischemia, hypoxia, oxidative stress and related pathologic processes ensue, further damaging vascular and neural function. One of the most important and specialized functions of the brain microvasculature is the blood-brain barrier (BBB), which controls the movement of molecules between blood circulation and the brain parenchyma. In this review, we are focusing on tight junctions (TJs), the multiprotein complexes that play an important role in establishing and maintaining barrier function. After a short introduction of the cell types that modulate barrier function via intercellular communication, we examine how age, age related pathologies and the aging of the immune system affects TJs. Then, we review how the TJs are affected in age associated neurodegenerative disorders: Alzheimer's disease and Parkinson's disease. Lastly, we summarize the TJ aspects of Huntington's disease and schizophrenia. Barrier dysfunction appears to be a common denominator in neurological disorders, warranting detailed research into the molecular mechanisms behind it. Learning the commonalities and differences in the pathomechanism of the BBB injury in different neurological disorders will predictably lead to development of new therapeutics that improve our life as we age.
Assuntos
Envelhecimento , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Microvasos/metabolismo , Doenças Neurodegenerativas/metabolismo , Junções Íntimas/metabolismo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/irrigação sanguínea , Humanos , Doença de Parkinson/metabolismoRESUMO
Recurrent spreading depolarizations occur in the cerebral cortex from minutes up to weeks following acute brain injury. Clinical evidence suggests that the immediate reduction of cerebral blood flow in response to spreading depolarization importantly contributes to lesion progression as the wave propagates over vulnerable tissue zones, characterized by potassium concentration already elevated prior to the passage of spreading depolarization. Here we demonstrate with two-photon microscopy in anesthetized mice that initial vasoconstriction in response to SD triggered experimentally with 1â¯M KCl is coincident in space and time with the large extracellular accumulation of potassium, as shown with a potassium indicator fluorescent dye. Moreover, pharmacological manipulations in combination with the use of potassium-sensitive microelectrodes suggest that large-conductance Ca2+-activated potassium (BK) channels and L-type voltage-gated calcium channels play significant roles in the marked initial vasoconstriction under elevated baseline potassium. We propose that potassium efflux through BK channels is a central component in the devastating neurovascular effects of spreading depolarizations in tissue at risk.
Assuntos
Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/fisiologia , Circulação Cerebrovascular/fisiologia , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Animais , Córtex Cerebral/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Indóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers.
Assuntos
Encéfalo/patologia , Endotélio/patologia , Melanoma/patologia , Metástase Neoplásica/patologia , Adulto , Barreira Hematoencefálica , Adesão Celular , Linhagem Celular Tumoral , Módulo de Elasticidade , Elasticidade , Humanos , Metástase Linfática/patologia , Masculino , Microscopia de Força Atômica , Invasividade Neoplásica , Estresse MecânicoRESUMO
Aging is associated with chronic inflammation partly mediated by increased levels of damage-associated molecular patterns, which activate pattern recognition receptors (PRRs) of the innate immune system. Furthermore, many aging-related disorders are associated with inflammation. PRRs, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), are expressed not only in cells of the innate immune system but also in other cells, including cells of the neurovascular unit and cerebral vasculature forming the blood-brain barrier. In this review, we summarize our present knowledge about the relationship between activation of PRRs expressed by cells of the neurovascular unit-blood-brain barrier, chronic inflammation, and aging-related pathologies of the brain. The most important damage-associated molecular pattern-sensing PRRs in the brain are TLR2, TLR4, and NLR family pyrin domain-containing protein-1 and pyrin domain-containing protein-3, which are activated during physiological and pathological aging in microglia, neurons, astrocytes, and possibly endothelial cells and pericytes.
Assuntos
Envelhecimento/metabolismo , Barreira Hematoencefálica/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Microvasos/metabolismo , Acoplamento Neurovascular , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Fatores Etários , Envelhecimento/imunologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/fisiopatologia , Humanos , Imunidade Inata , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/fisiopatologia , Microvasos/imunologia , Microvasos/fisiopatologia , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.
Assuntos
Células Endoteliais/citologia , Melanoma , Análise de Célula Única/métodos , Adulto , Fenômenos Biomecânicos , Barreira Hematoencefálica , Encéfalo/citologia , Encéfalo/patologia , Adesão Celular , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Humanos , Masculino , Microscopia de Força AtômicaRESUMO
Cerebral pericytes are mural cells embedded in the basement membrane of capillaries. Increasing evidence suggests that they play important role in controlling neurovascular functions, i.e. cerebral blood flow, angiogenesis and permeability of the blood-brain barrier. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. We detected expression of NOD1, NOD2, NLRC5, NLRP1-3, NLRP5, NLRP9, NLRP10 and NLRX mRNA in non-treated cells. Among the ten known human TLRs, TLR2, TLR4, TLR5, TLR6 and TLR10 were found to be expressed. Inflammatory mediators induced the expression of NLRA, NLRC4 and TLR9 and increased the levels of NOD2, TLR2, inflammasome-forming caspases and inflammasome-cleaved interleukins. Oxidative stress, on the other hand, upregulated expression of TLR10 and NLRP9. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1ß. TNF-α and IFN-γ increased the levels of pro-IL-1ß and pro-caspase-1 proteins; however, no canonical activation of NLRP1, NLRP2, NLRP3 or NLRC4 inflammasomes could be observed in human brain vascular pericytes. On the other hand, we could demonstrate secretion of active IL-1ß in response to non-canonical inflammasome activation, i.e. intracellular LPS or infection with E. coli bacteria. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation.
Assuntos
Encéfalo/metabolismo , Inflamassomos/metabolismo , Pericitos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/metabolismo , Transdução de SinaisRESUMO
Endothelial-mesenchymal transition (EndMT) is an important mechanism during organ development and in certain pathological conditions. For example, EndMT contributes to myofibroblast formation during organ fibrosis, and it has been identified as an important source of cancer-associated fibroblasts, facilitating tumor progression. Recently, EndMT was proposed to modulate endothelial function during intravasation and extravasation of metastatic tumor cells. Evidence suggests that endothelial cells are not passive actors during transendothelial migration (TEM) of cancer cells, as there are profound changes in endothelial junctional protein expression, signaling, permeability, and contractility. This review describes these alterations in endothelial characteristics during TEM of metastatic tumor cells and discusses them in the context of EndMT. EndMT could play an important role during metastatic intravasation and extravasation, a novel hypothesis that may lead to new therapeutic approaches to tackle metastatic disease.
Assuntos
Células Endoteliais/patologia , Transição Epitelial-Mesenquimal , Neoplasias/patologia , Migração Transendotelial e Transepitelial , Animais , Comunicação Celular , Células Endoteliais/metabolismo , Humanos , Metástase Neoplásica , Neoplasias/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain.
Assuntos
Barreira Hematoencefálica/metabolismo , Moléculas de Adesão Celular/metabolismo , Córtex Cerebral/metabolismo , Substância Cinzenta/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Proteínas de Junções Íntimas/metabolismo , Substância Branca/metabolismo , Animais , Astrócitos/metabolismo , Simulação por Computador , Feminino , Humanos , Masculino , Camundongos , Estrutura Molecular , Pericitos/metabolismo , Suínos , Junções Íntimas/metabolismoRESUMO
Cerebral endothelial cells (CECs) forming the blood-brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen- and damage-associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD-like receptors (NLRs) - including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX - in human brain endothelial cells. Inflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and IL-1ß had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase-cleaved interleukins IL-1ß and IL-33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL-1ß secretion in a caspase- and ERK1/2 kinase-dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood-brain barrier in neuroimmune and neuroinflammatory processes.
Assuntos
Encéfalo/metabolismo , Células Endoteliais/metabolismo , Inflamassomos/metabolismo , Proteína Adaptadora de Sinalização NOD1/fisiologia , Proteína Adaptadora de Sinalização NOD2/fisiologia , Animais , Células Cultivadas , Humanos , RatosRESUMO
The most important obstacle to the drug delivery into the brain is the presence of the blood-brain barrier, which limits the traffic of substances between the blood and the nervous tissue. Therefore, adequate in vitro models need to be developed in order to characterize the penetration properties of drug candidates into the central nervous system. This review article summarizes the presently used and the most promising in vitro BBB models based on the culture of brain endothelial cells. Robust models can be obtained using primary porcine brain endothelial cells and rodent coculture models, which have low paracellular permeability and express functional efflux transporters, showing good correlation of drug penetration data with in vivo results. Models mimicking the in vivo anatomophysiological complexity of the BBB are also available, including triple coculture (culture of brain endothelial cells in the presence of pericytes and astrocytes), dynamic, and microfluidic models; however, these are not suitable for rapid, high throughput studies. Potent human cell lines would be needed for easily available and reproducible models which avoid interspecies differences.
Assuntos
Barreira Hematoencefálica/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Humanos , Técnicas In Vitro/métodos , Modelos Biológicos , PermeabilidadeRESUMO
Two new arylbenzofuran-type neolignans, 1"-dehydroegonol 3"-methyl ether (1) and egonol 3"-methyl ether (2), and four known lignan derivatives, namely, helioxanthin (3), (7E)-7,8-dehydroheliobuphthalmin (4), heliobuphthalmin (5), and 7-acetoxyhinokinin (6), were isolated from a chloroform-soluble partition of the methanol extract of the fresh roots of Heliopsis helianthoides var. scabra. These six compounds were evaluated in vitro in terms of their ability to inhibit the various steps involved in brain tumor metastasis formation. Compounds 3 and 4 inhibited the migration of both melanoma and brain endothelial cells, and 3 also reduced the adhesion of melanoma cells to the brain endothelium. Furthermore, 3 and 4 additionally enhanced the barrier function of the blood-brain barrier and the expression of the tight junction protein ZO-1 at the junctions of the endothelial cells. These findings suggest that 3 and 4 may have the potential to interfere with different steps of brain metastasis formation and to enhance the barrier function of cerebral endothelial cells.
Assuntos
Asteraceae/química , Encéfalo/efeitos dos fármacos , Lignanas/isolamento & purificação , Lignanas/farmacologia , Citoesqueleto de Actina , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Hungria , Lignanas/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Raízes de Plantas/química , Ratos , Proteína da Zônula de Oclusão-1/efeitos dos fármacosRESUMO
During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.